This article is from Journal of Neuroinflammation, volume 11.

Abstract

Background: Repetitive hypoxic preconditioning (RHP) creates an anti-inflammatory phenotype that protects from stroke-induced injury for months after a 2-week treatment. The mechanisms underlying long-term tolerance are unknown, though one exposure to hypoxia significantly increased peripheral B cell representation. For this study, we sought to determine if RHP specifically recruited B cells into the protected ischemic hemisphere, and whether RHP could phenotypically alter B cells prior to stroke onset. Methods: Adult, male SW/ND4 mice received RHP (nine exposures over 2 weeks; 8 to 11 % O2; 2 to 4 hours) or identical exposures to 21 % O2 as control. Two weeks following RHP, a 60-minute transient middle cerebral artery occlusion was induced. Standard techniques quantified CXCL13 mRNA and protein expression. Two days after stroke, leukocytes were isolated from brain tissue (70:30 discontinuous Percoll gradient) and profiled on a BD-FACS Aria flow cytometer. In a separate cohort without stroke, sorted splenic CD19+ B cells were isolated 2 weeks after RHP and analyzed on an Illumina MouseWG-6 V2 Bead Chip. Final gene pathways were determined using Ingenuity Pathway Analysis. Student’s t-test or one-way analysis of variance determined significance (P < 0.05). Results: CXCL13, a B cell-specific chemokine, was upregulated in post-stroke cortical vessels of both groups. In the ischemic hemisphere, RHP increased B cell representation by attenuating the diapedesis of monocyte, macrophage, neutrophil and T cells, to quantities indistinguishable from the uninjured, contralateral hemisphere. Pre-stroke splenic B cells isolated from RHP-treated mice had >1,900 genes differentially expressed by microarray analysis. Genes related to B-T cell interactions, including antigen presentation, B cell differentiation and antibody production, were profoundly downregulated. Maturation and activation were arrested in a cohort of B cells from pre-stroke RHP-treated mice while regulatory B cells, a subset implicated in neurovascular protection from stroke, were upregulated. Conclusions: Collectively, our data characterize an endogenous neuroprotective phenotype that utilizes adaptive immune mechanisms pre-stroke to protect the brain from injury post-stroke. Future studies to validate the role of B cells in minimizing injury and promoting central nervous system recovery, and to determine whether B cells mediate an adaptive immunity to systemic hypoxia that protects from subsequent stroke, are needed.

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